Various publications or patents are referred to in parentheses throughout this application to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein. Complete citations of scientific publications are set forth in the text or at the end of the specification.
Bone Marrow (BM) is the major source of both lymphocytes (immune cells) and erythrocytes in the adult. Among the various cells that constitute the BM are primitive hematopoietic pluripotent stem cells and progenitor cells. An important property of stem cells is their ability to both proliferate, which ensures a continuous supply throughout the lifetime of an individual, and differentiate into the mature cells of the peripheral blood system. When necessary, a pluripotent stem cell can begin to differentiate, and after successive divisions become committed, thus losing the capacity for self-renewal, to a particular line of development. All of the circulating blood cells, including erythrocytes, leukocytes or lymphocytes, granulocytes and platelets originate from various progenitor cells that are themselves derived from precursor stem cells.
The morphologically recognizable and functionally capable cells circulating in the blood include erythrocytes (red blood cells), leukocytes (white blood cells including both B and T cells), non B- and T-lymphocytes, phagocytes, neutrophilic, eosinophilic and basophilic granulocytes, and platelets. These mature cells are derived, on demand, from dividing progenitor cells, such as erythroblasts (for erythrocytes), lymphoid precursors, myeloblasts (for phagocytes including monocytes, macrophages and neutrophils), promyelocytes and myelocytes (for the various granulocytes) and megakaryocytes for the platelets. As stated above, these progenitor cells are themselves derived from precursor stem cells.
A complex network of soluble factors as well as inter- and intra-cellular interactions regulate the proliferation and differentiation of a finite pool of hematopoietic stem cells. Proliferation and differentiation of hematopoietic cells are regulated by hormone-like growth and differentiation factors designated as colony-stimulating factors (CSF) (Metcalf, D. Nature 339, 27-30 (1989)). CSF can be classified into several factors according to the stage of the hematopoietic cells to be stimulated and the surrounding conditions as follows: granulocyte colony-stimulation factor (G-CSF), granulocyte-macrophage colony-stimulation factor (GM-CSF), macrophage colony-stimulation factor (M-CSF), and interleukin 3 (IL-3).
Small amounts of certain hematopoietic growth factors account for the differentiation of stem cells into a variety of blood cell progenitors, for the tremendous proliferation of those cells, and for their differentiation into mature blood cells. For instance, G-CSF participates greatly in the differentiation and growth of neutrophilic granulocytes and plays an important role in the regulation of blood levels of neutrophils and the activation of mature neutrophils (Nagata, S., “Handbook of Experimental Pharmacology”, volume “Peptide Growth Factors and Their Receptors”, eds. Sporn, M. B. and Roberts, A. B., Spring-Verlag, Heidelberg, Vol.95/1, pp.699-722 (1990); Nicola, N. A. et al., Annu. Rev. Biochem. 58, pp.45-77 (1989)). It is also reported that G-CSF stimulates the growth of tumor cells such as myeloid leukemia cells. (Nicola and Metcalf, Proc. Natl. Acad. Sci. USA, 81, 3765-3769 (1984); Begley et al., Leukemia, 1, 1-8 (1987).) Other growth factors include, erythropoietin (EPO), which is responsible for stimulating the differentiation of erythroblasts into erythrocytes and Macrophage-Colony Stimulating Factor (M-CSF) responsible for stimulating the differentiation of myeloblasts and myelocytes into monocytes.
Growth factors are part of a family of chemical messengers known as the cytokines. Cytokines are among the factors that act upon the hematopoietic system to regulate blood cell proliferation and differentiation. Cytokines are also important mediators of the immune response being secreted by both B and T cells, as well as other various lymphocytes. Cytokines encourage cell growth, promote cell activation, direct cellular traffic, act as messengers between cells of the hematopoietic system, and destroy target cells (i.e., cancer cells). Among the various components involved in the modulation and regulation of the hematopoietic system that cytokines play a role in modulating are the tachykinins.
The tachykinins are immune and hematopoietic modulators that belong to a family of peptides encoded by the preprotachykinin-I (PPT-1) gene (1). The tachykinins can be released in the BM and other lymphoid organs as neurotransmitters or from the resident BM immune cells (2-6). In the BM, PPT-I and other hematopoietic growth factors regulate expression of each other through autocrine and paracrine activities. It is believed that various cytokines induce the expression of the PPT-I gene in BM mesenchymal cells (2). The tachykinin family of peptides exerts pleiotropic functions such as neurotransmission and immune/hematopoietic modulation.
PPT-1 peptides exert both stimulatory and inhibitory hematopoietic effects by interacting with different affinities to the G-protein coupled receptors: NK-1, NK-2 and NK-3 (7). NK-1 and NK-2 expression has been reported in BM cells (8). NK-1 is induced in BM cells by cytokines and other stimulatory hematopoietic regulators. NK-2 is constitutively expressed in BM cells that are unstimulated or stimulated with suppressive hematopoietic regulators. NK-1 and NK-2 are not co-expressed in BM cells because NK-1 induction by cytokines is correlated with the down regulation of NK-2. In BM cells, NK-1 expression requires cell stimulation whereas its expression in neural tissue is constitutive (2, 9, 10). It is believed that a particular cytokine discriminates between the expression of NK-1 and NK-2, which directs the type of BM functions: stimulatory vs. inhibitory (8).
Substance P (SP), the major tachykinin released in the BM, stimulates hematopoiesis through interactions with the neurokinin-1 (NK-1) receptor, which is resident on BM stroma, immune cells and other lymphoid organ cells. Hence, the expression of NK-1 determines the hematopoietic response of the tachykinins. NK-2 inhibits hematopoiesis by interacting with neurokinin-A, another tachykinin encoded for by the PPT-I gene. Recently the present inventors have discovered that the stimulatory effects mediated by NK-1 can be changed to hematopoietic inhibition in the presence of the amino terminal of SP, a fragment found endogenously in the BM due to enzymatic digestion of SP by endogenous endopeptidases. Further, dysregulated expression of the PPT-1 gene has been associated with different pathologies such as cancer (Bost et al., 1992b; Henning et al, 1995; Ho et al., 1996; Michaels, 1998; Rameshwar et al, 1997a).
Under normal circumstances, the BM is able to respond quickly to an increased demand for a particular type of cell. The pluripotential stem cell is capable of creating and reconstituting all the cells that circulate in the blood, including both red and white blood cells and platelets. As stated, progenitor cells that derive from stem cells can replicate and differentiate at an astounding, if not alarming rate. On average, 3-10 billion lymphocyte cells can be generated in an hour. The BM can increase this by 10 fold in response to need. However, in the throes of a diseased state, the BM may not produce enough stem cells, may produce too many stem cells or various ones produced may begin to proliferate uncontrollably. Further complications arise when these stem cells or their associated progenitor are not able to differentiate into the various morphologically recognizable and functionally capable cells circulating in the blood.
Lymphoproliferative syndromes consist of types of diseases known as leukemia and malignant lymphoma, which can further be classified as acute and chronic myeloid or lymphocytic leukemia, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. These diseases are characterized by the uncontrollable multiplication or proliferation of leukocytes (primarily the B-cells) and tissue of the lymphatic system, especially lymphocyte cells produced in the BM and lymph nodes.
Lymphocytes (also called leukocytes) are core components of the body's immune system, which is one of the principal mechanisms by which the body attacks and controls cancers. Lymphocytes, or their derivatives, recognize the foreign antigenic nature of cancer cells or of antibodies associated therewith and attack the cancer cells. Upon exposure to a foreign antigen in the human body, lymphoctes naturally proliferate or multiply to combat the antigen.
There are two broad sub-types of lymphocyte cells. These are known as B and T cells. All of them are derived from the bone marrow but T cells undergo a process of maturation in the thymus gland. Mature lymphocytes all have a similar appearance. They are small cells with a deeply basophilic nucleus and scanty cytoplasm. B and T cells circulate in the blood and through body tissues. B cells primarily work by secreting soluble substances called antibodies. Each B cell is programmed to make one specific antibody. When a B cell encounters its triggering anitgen, it goes through a process wherein it is changed into many large plasma cells. Hence, B cells give rise to plasma cells which secrete a specific immunoglobulin (antibodies). T cells also respond to antigens. Some of them (CD4+) secrete lymphokines that act on other cells, thus regulating the complex workings of the immune response. Others (CD8+, cytotoxic) directly contact infected cells and are able to cause lysis therby destroying the infected cells.
Leukemia and other such B-cell malignancies, such as acute and chronic myeloid and lymphocytic leukemia as well as the B-cell subtype of Hodgkins and non-Hodgkin's lymphoma, are examples of lymphoproliferative syndromes that are significant contributors to cancer mortality. In fact, the majority of chronic lymphocytic leukemias are of B-cell lineage. Freedman, Hematol. Oncol. Clin. North Am. 4:405 (1990).
Leukemia can be defined as the uncontrolled proliferation of a clone of abnormal hematopoietic cells. Leukemias are further typically characterized as being myelocytic or lymphocytic. Myeloid leukemias affect the descendents of the myeloid lineage, where as the lymphocytic leukemias involve abnormalities in the lymphoid lineage. Most B cell leukemias and lymphomas are monoclonal, meaning that all of the related tumor cells are derived from one particular aberrant cell.
Generally, leukemia is a neoplastic disease in which white corpuscle maturation is arrested at a primitive stage of cell development. The disease is characterized by an increased number of leukemic blast cells in the bone marrow and by varying degrees of failure to produce normal hematopoietic cells. The condition may be either acute or chronic. Acute myelocytic leukemia (AML) arises from bone marrow hematopoietic stem cells or their progeny. The term “acute myelocytic leukemia” subsumes several subtypes of leukemia e.g. myeloblastic leukemia, promyelocytic leukemia and myelomonocytic leukemia and is a form of cancer that affects the cells producing myeloid blood cells in the BM. As stated above, myeloid cells are red blood cells, platelets and all white blood cells (which include: neutrophils, monocytes, macrophages, eosinophils and basophils). Primarily, AML involves abnormal white blood cells of the neutrophil type. Production of blood cells is obstructed and immature cells known as “blast cells” accumulate in the bone marrow. These cells are unable to mature and differentiate properly leading to a significant reduction of normal blood cells in the circulation. The accumulation of blast cells in the BM prevents production of other cell types resulting in anemia and low platelet blood counts. Acute lymphocytic leukemia (ALL) arises in lymphoid tissues and ordinarily first manifests its presence in bone marrow. ALL is primarily a form of cancer that affects the lymphocytes and lymphocyte producing cells in the BM.
Chronic myelogenous leukemia (CML) is characterized by abnormal proliferation of immature granulocytes, for example, neutrophils, eosinophils and basophils, in the blood, bone marrow, the spleen, liver and sometimes in other tissues. A large portion of chronic myelogenous leukemia patients develop a transformation into a pattern indistinguishable from the acute form of the disease.
This change is known as the “blast crises”. Chronic lymphocytic leukemia (CLL) is a form of leukemia in which there is an excess number of mature, but poorly functioning lymphocytes in the circulating blood. It is to be noted that the rate of production of lymphocytes is not significantly increased and may in fact even be slower than normal. CLL has several phases. In the early phase it characterized by the accumulation of small mature functionally-incompetent malignant B-cells having a lengthened life span. The late stages of CLL are characterized by significant anemia and/or thrombocytopenia.
The two main types of lymphoma are Hodgkin's and non-Hodgkin's lymphoma. Hodgkin's disease is a cancer of the lymphatic system—the network of lymph glands and channels that occurs throughout the body. The defining feature of Hogkin's disease is the presence of a distinctive abnormal lymphocyte called a Reed-Sternberg cell. There are five recognized sub-groups of Hodgkin's disease; these are: lymphocyte rich, nodular sclerosing, mixed cellularity, lymphocyte depleted and nodular lymphocyte predominant (which predominantly affects one isolated lymph node). All other types of lymphoma are collectively known as non-Hodgkin's lymphoma. There are thirty sub-types of non-Hodgkin's type lymphoma.
Traditional methods of treating these B-cell malignancies, which includes chemotherapy and radiotherapy, have limited utility due to toxic side effects. Short-term side effects of chemotherapy may include significant toxicity, extreme nausea, vomiting, and serious discomfort. The long-term side effects may include diabetes, other forms of B-cell malignancies, other forms of cancer, heart, lung or other organ disease, fatal bleeding during remission induction, and myelodysplasia. The short-term side effects of radiotherapy may include extreme nausea, vomiting, serious discomfort, sterility and infertility. The long-term side effects of radiotherapy may include other forms B-cell malignancies, cancer, thyroid gland, spleen or other organ failure. These side effects may be moderated by reduced dosages, however, that increases the risk of remission.
Another traditional method for treating B-cell malignancies includes either BM or stem cell transplantation. However, these procedures are plagued with exorbitant cost and high rates of failure. It is both difficult and costly to locate a sufficient donor and even when one is located, rejection of the transplanted cells often takes place, which in turn can lead to graft versus host disease. Most often, these treatments also include a combination of both chemo and radiotherapies, hence, the concomitant risks involved therein would apply here as well.
There is, therefore, a need for a more non-evasive treatment for lymphoproliferative diseases related to either an increase or decrease in differentiation, as well as uncontrolled proliferation. The present invention involves a novel gene, its antisense polynucleotide sequence, the coded for protein and antibodies immunospecific to the coded for protein. More specifically, the present invention provides pharmaceutical compositions of the novel gene, its antisense sequence, the protein and/or antibodies immunospecific to the protein, that can be used to either increase or decrease lymphocyte differentiation and may be useful in inhibiting white blood cell proliferation.
Hence, the methods of the present invention are useful for the prevention and treatment of lymphoproliferative syndromes such as B-cell related maladies, including but not limited to acute and chronic myeloid and lymphocytic leukemia as well as the B-cell subtype of Hodgkin's and non-Hodgkin's lymphomas. Further, the methods of the present invention can be used to increase the effectiveness of both chemo- and radiotherapy. Further still, the use of monoclonal antibodies, in conjunction with the gene, antisense polynucleotide or protein of the present invention, to direct radionuclides, toxins, or other therapeutic agents offers the possibility that such agents can be delivered at lower dosages, selectively to tumor sites, thus limiting toxicity to normal tissues.